Genomic view of heavy-ion-induced deletions associated with distribution of essential genes in Arabidopsis thaliana.

Heavy-ion beam, a type of ionizing radiation, has been applied to plant breeding as a powerful mutagen and is a promising tool to induce large deletions and chromosomal rearrangements. The effectiveness of heavy-ion irradiation can be explained by linear energy transfer (LET; keV µm-1). Heavy-ion beams with different LET values induce different types and sizes of mutations. It has been suggested that deletion size increases with increasing LET value, and complex chromosomal rearrangements are induced in higher LET radiations. In this study, we mapped heavy-ion beam-induced deletions detected in Arabidopsis mutants to its genome. We revealed that deletion sizes were similar between different LETs (100 to 290 keV µm-1), that their upper limit was affected by the distribution of essential genes, and that the detected chromosomal rearrangements avoid disrupting the essential genes. We also focused on tandemly arrayed genes (TAGs), where two or more homologous genes are adjacent to one another in the genome. Our results suggested that 100 keV µm-1 of LET is enough to disrupt TAGs and that the distribution of essential genes strongly affects the heritability of mutations overlapping them. Our results provide a genomic view of large deletion inductions in the Arabidopsis genome.

Evolution of sex-determination in dioecious plants: From active Y to X/A balance?

Sex chromosomes in plants have been known for a century, but only recently have we begun to understand the mechanisms behind sex determination in dioecious plants. Here, we discuss evolution of sex determination, focusing on Silene latifolia, where evolution of separate sexes is consistent with the classic "two mutations" model-a loss of function male sterility mutation and a gain of function gynoecium suppression mutation, which turned an ancestral hermaphroditic population into separate males and females. Interestingly, the gynoecium suppression function in S. latifolia evolved via loss of function in at least two sex-linked genes and works via gene dosage balance between sex-linked, and autosomal genes. This system resembles X/A-ratio-based sex determination systems in Drosophila and Rumex, and could represent a steppingstone in the evolution of X/A-ratio-based sex determination from an active Y system.

The origin and evolution of sex chromosomes, revealed by sequencing of the Silene latifolia female genome.

White campion (Silene latifolia, Caryophyllaceae) was the first vascular plant where sex chromosomes were discovered. This species is a classic model for studies on plant sex chromosomes due to presence of large, clearly distinguishable X and Y chromosomes that originated de novo about 11 million years ago (mya), but lack of genomic resources for this relatively large genome (∼2.8 Gb) remains a significant hurdle. Here we report S. latifolia female genome assembly integrated with sex-specific genetic maps of this species, focusing on sex chromosomes and their evolution. The analysis reveals a highly heterogeneous recombination landscape with strong reduction in recombination rate in the central parts of all chromosomes. Recombination on the X chromosome in female meiosis primarily occurs at the very ends, and over 85% of the X chromosome length is located in a massive (∼330 Mb) gene-poor, rarely recombining pericentromeric region (Xpr). The results indicate that the non-recombining region on the Y chromosome (NRY) initially evolved in a relatively small (∼15 Mb), actively recombining region at the end of the q-arm, possibly as a result of inversion on the nascent X chromosome. The NRY expanded about 6 mya via linkage between the Xpr and the sex-determining region, which may have been caused by expanding pericentromeric recombination suppression on the X chromosome. These findings shed light on the origin of sex chromosomes in S. latifolia and yield genomic resources to assist ongoing and future investigations into sex chromosome evolution.

A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.

How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic "two-factor" model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males.

Femoral Valgus Correction Angle for the Intramedullary Alignment Rod Is Strongly Associated with Femoral Lateral Bowing in Japanese Patients with Varus Knee Osteoarthritis Undergoing Total Knee Arthroplasty.

Background: This study aimed to investigate factors, such as differences in femoral shape, that could affect the femoral valgus correction angle (VCA) for the intramedullary alignment rod (IM rod) by using a three-dimensional (3D) measurement system in patients with varus knee osteoarthritis undergoing total knee arthroplasty (TKA). Methods: A total of 305 knees in 233 Japanese patients with varus knee osteoarthritis who underwent primary TKA by using Jig Engaged 3D Pre-Operative Planning Software for the TKA operation support system was examined. We retrospectively analysed factors, such as the shape of the proximal, middle, and distal femur in the coronal plane, all of which could affect the VCA for the IM rod, by multiple linear regression analyses. Results: The VCA for the IM rod was 5.9° ± 1.6° (range: 1.7° to 10.7°), and the femoral lateral bowing angle (FBA) was 3.5° ± 3.2°. Major factors independently associated with the VCA for the IM rod were the FBA (ß: 0.75), femoral offset (ß: 0.38), and the medial angle between the mechanical femoral axis and the line that connects the distal margins of the medial and lateral femoral condyles (ß: -0.16). The model was created by stepwise multiple linear regression (F = 266.6, p < 0.001, and estimated effect size = 4.4) explained 85% of the variance in the VCA for the IM rod (R 2 = 0.85). Conclusions: The VCA for the IM rod was most strongly associated with femoral lateral bowing in patients with varus knee osteoarthritis undergoing TKA. Our findings suggest that preoperatively measuring the VCA for the IM rod in patients with femoral lateral bowing by using a 3D measurement system could be useful for accurate coronal alignment of the femoral component in TKA.

Evaluation of an operation support system using the femoral anterior tangent line to determine intraoperative femoral component rotation in total knee arthroplasty.

BACKGROUND: The femoral anterior tangent (FAT) line refers to a line parallel to the anterior surface of the distal femur in the axial plane. This study aimed to evaluate the effectiveness of a new operation support system which uses the FAT line to set the femoral component rotational alignment in total knee arthroplasty (TKA). METHODS: A total of 170 consecutive knees in 139 patients undergoing primary TKA with the JIGEN (Jig Engaged Three-dimensional (3D) Pre-Operative Planning Software for TKA) operation support system was examined. The JIGEN system creates 3D models of bones using computed tomography data, allowing for surgical simulations such as positioning of implants while calculating positions of the intramedullary alignment rod (IM rod) and surgical jig. We retrospectively analyzed the FAT line angle relative to the surgical epicondylar axis (SEA) on the axis plane perpendicular to the IM rod and evaluated the accuracy of the femoral component alignment after TKA with the 3D measurement system. RESULTS: The FAT line was 9.6° ± 3.7° (range, 1.4°-20.4°) internally rotated relative to the SEA. The average absolute error was 1.4° ± 1.1° in the coronal plane, 2.0° ± 1.5° in the sagittal plane, and 1.6° ± 1.3° in the axial plane. The femoral component outliers (i.e., >3° away from the goal alignment) were 7.7% in the coronal plane, 20.6% in the sagittal plane, and 10.3% in the axial plane. CONCLUSIONS: Our findings suggest that the FAT line is a reliable and reproducibly identifiable axis for the accurate determination of proper rotational alignment in TKA. An operation support system which uses the FAT line for determining intraoperative femoral component rotation can effectively achieve highly accurate positioning of the femoral component in TKA.

Double Mutant Analysis with the Large Flower Mutant, ohbana1, to Explore the Regulatory Network Controlling the Flower and Seed Sizes in Arabidopsis thaliana.

Two growth processes, cell proliferation and expansion, determine plant species-specific organ sizes. A large flower mutant in Arabidopsis thaliana, ohbana1 (ohb1), was isolated from a mutant library. In the ohb1 flowers, post-mitotic cell expansion and endoreduplication of nuclear DNA were promoted. The whole-genome resequencing and genetic analysis results showed that the loss of function in MEDIATOR16 (MED16), a mediator complex subunit, was responsible for the large flower phenotypes exhibited by ohb1. A phenotypic analysis of the mutant alleles in MED16 and the double mutants created by crossing ohb1 with representative large flower mutants revealed that MED16 and MED25 share part of the negative petal size regulatory pathways. Furthermore, the double mutant analyses suggested that there were genetically independent pathways leading to cell size restrictions in the floral organs which were not related to the MED complex. Several double mutants also formed larger and heavier seeds than the wild type and single mutant plants, which indicated that MED16 was involved in seed size regulation. This study has revealed part of the size-regulatory network in flowers and seeds through analysis of the ohb1 mutant, and that the size-regulation pathways are partially different between floral organs and seeds.

Arabidopsis EGY1 Is Critical for Chloroplast Development in Leaf Epidermal Guard Cells.

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two 40Ar17+ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells.

Arterial embolism due to massive cement leakage during total hip arthroplasty revision.

Total hip arthroplasty with cementing techniques leads to good clinical outcomes, but critical vascular complications can sometimes occur due to cement leakage into the pelvis. In this report, we describe a case of massive cement leakage that caused an arterial embolism. When exfoliating cement from an artery, the surgeon should note not only direct injury to the vessels but also the potential for arterial embolism.

TGD5 is required for normal morphogenesis of non-mesophyll plastids, but not mesophyll chloroplasts, in Arabidopsis.

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.

An Argon-Ion-Induced Pale Green Mutant of Arabidopsis Exhibiting Rapid Disassembly of Mesophyll Chloroplast Grana.

Argon-ion beam is an effective mutagen capable of inducing a variety of mutation types. In this study, an argon ion-induced pale green mutant of Arabidopsis thaliana was isolated and characterized. The mutant, designated Ar50-33-pg1, exhibited moderate defects of growth and greening and exhibited rapid chlorosis in photosynthetic tissues. Fluorescence microscopy confirmed that mesophyll chloroplasts underwent substantial shrinkage during the chlorotic process. Genetic and whole-genome resequencing analyses revealed that Ar50-33-pg1 contained a large 940 kb deletion in chromosome V that encompassed more than 100 annotated genes, including 41 protein-coding genes such as TYRAAt1/TyrA1, EGY1, and MBD12. One of the deleted genes, EGY1, for a thylakoid membrane-localized metalloprotease, was the major contributory gene responsible for the pale mutant phenotype. Both an egy1 mutant and F1 progeny of an Ar50-33-pg1 × egy1 cross-exhibited chlorotic phenotypes similar to those of Ar50-33-pg1. Furthermore, ultrastructural analysis of mesophyll cells revealed that Ar50-33-pg1 and egy1 initially developed wild type-like chloroplasts, but these were rapidly disassembled, resulting in thylakoid disorganization and fragmentation, as well as plastoglobule accumulation, as terminal phenotypes. Together, these data support the utility of heavy-ion mutagenesis for plant genetic analysis and highlight the importance of EGY1 in the structural maintenance of grana in mesophyll chloroplasts.

A Method Enabling Comprehensive Isolation of Arabidopsis Mutants Exhibiting Unusual Root Mechanical Behavior.

Root penetration into soils is fundamental for land plants to support their own aboveground parts and forage water and nutrients. To elucidate the molecular mechanisms underlying root mechanical penetration, mutants defective in this behavior need to be comprehensively isolated; however, established methods are currently scarce. We herein report a method to screen for these mutants of Arabidopsis thaliana and present their phenotypes. We isolated five mutants using this method, tentatively named creep1 to creep5, the primary roots of which crept over the surface of horizontal hard medium that hampered penetration by the primary root of the wild type, thereby forcing it to spring up on the surface and die. By examining root skewing, which is induced by a touch stimulation that is generated as the primary roots grow along a vertical impenetrable surface, the five creep mutants were subdivided into three groups, namely mutants with the primary root skewing leftward, those skewing rightward, and that growing dispersedly. While the majority of wild type primary roots skewed slightly leftward, nearly half of the primary roots of creep1 and creep5 skewed rightward as viewed from above. The primary roots of creep4 displayed scattered growth, while those of creep2 and creep3 showed a similar phenotype to the wild type primary roots. These results demonstrate the potential of the method developed herein to isolate various mutants that will be useful for investigating root mechanical behavior regulation not only in Arabidopsis, but also in major crops with economical value.

Isolation and characterization of a motility-defective mutant of Euglena gracilis.

Euglena gracilis is a green photosynthetic microalga that swims using its flagellum. This species has been used as a model organism for over half a century to study its metabolism and the mechanisms of its behavior. The development of mass-cultivation technology has led to E. gracilis application as a feedstock in various products such as foods. Therefore, breeding of E. gracilis has been attempted to improve the productivity of this feedstock for potential industrial applications. For this purpose, a characteristic that preserves the microalgal energy e.g., reduces motility, should be added to the cultivars. The objective of this study was to verify our hypothesis that E. gracilis locomotion-defective mutants are suitable for industrial applications because they save the energy required for locomotion. To test this hypothesis, we screened for E. gracilis mutants from Fe-ion-irradiated cell suspensions and established a mutant strain, M 3 - ZFeL, which shows defects in flagellum formation and locomotion. The mutant strain exhibits a growth rate comparable to that of the wild type when cultured under autotrophic conditions, but had a slightly slower growth under heterotrophic conditions. It also stores 1.6 times the amount of paramylon, a crystal of ß-1,3-glucan, under autotrophic culture conditions, and shows a faster sedimentation compared with that of the wild type, because of the deficiency in mobility and probably the high amount of paramylon accumulation. Such characteristics make E. gracilis mutant cells suitable for cost-effective mass cultivation and harvesting.

Sexual Difference in the Optimum Environmental Conditions for Growth and Maturation of the Brown Alga Undaria pinnatifida in the Gametophyte Stage.

Undaria pinnatifida is an annual brown kelp growing naturally in coastal areas as a major primary producer in temperate regions and is cultivated on an industrial scale. Kelps have a heteromorphic life cycle characterized by a macroscopic sporophyte and microscopic sexual gametophytes. The sex-dependent effects of different environmental factors on the growth and maturation characteristics of the gametophyte stage were investigated using response surface methodology. Gametophytes were taken from three sites in Japan: Iwate Prefecture, Tokushima Prefecture, and Kagoshima Prefecture in order to confirm the sexual differences in three independent lines. Optimum temperature and light intensity were higher for males (20.7-20.9 °C and 28.6-33.7 µmol m-2 s-1, respectively) than females (16.5-19.8 °C and 26.9-32.5 µmol m-2 s-1), and maturity progressed more quickly in males than females. Optimum wavelengths of light for growth and maturation of the gametophytes were observed for both blue (400-500 nm, λmax 453 nm) and green (500-600 nm; λmax 525 nm) lights and were sex-independent. These characteristics were consistent among the three regional lines. Slower growth optima and progress of maturation could be important for female gametophytes to restrict fertilization and sporophyte germination to the lower water temperatures of autumn and winter, and suggest that the female gametophyte may be more sensitive to temperature than the male. The sexual differences in sensitivity to environmental factors improved the synchronicity of sporeling production.

An asexual flower of Silene latifolia and Microbotryum lychnidis-dioicae promotes sex-organ development.

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae.

Immediate Dosage Compensation Is Triggered by the Deletion of Y-Linked Genes in Silene latifolia.

The loss of functional genes from non-recombining sex-specific chromosomes [1, 2], such as the Y chromosomes in mammals [3] or W chromosomes in birds [4], should result in an imbalance of gene products for sex-linked genes [5]. Different chromosome-wide systems that rebalance gene expression are known to operate in organisms with relatively old sex chromosomes [6]; e.g., Drosophila overexpress X-linked genes in males [7], while mammals shut down one of the X chromosomes in females [8]. It is not known how long it takes for a chromosome-wide dosage compensation system to evolve. To shed light on the early evolution of dosage compensation, we constructed a high-density Y-deletion map and used deletion mutants to manipulate gene dose and analyze gene expression in white campion (Silene latifolia), which evolved dioecy and sex chromosomes only 11 million years ago [9]. We demonstrate that immediate dosage compensation can be triggered by deletions in a large portion of the p arm of the Y chromosome. Our results indicate that dosage compensation in S. latifolia does not have to evolve gene by gene because a system to upregulate gene expression is already operating on part of the X chromosome, which likely represents an intermediate step in the evolution of a chromosome-wide dosage compensation system in this species.

Development of the VIGS System in the Dioecious Plant Silene latifolia.

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.

Arabidopsis PARC6 Is Critical for Plastid Morphogenesis in Pavement, Trichome, and Guard Cells in Leaf Epidermis.

Recently, a recessive Arabidopsis thaliana mutant with abundant stromules in leaf epidermal pavement cells was visually screened and isolated. The gene responsible for this mutant phenotype was identified as PARC6, a chloroplast division site regulator gene. The mutant allele parc6-5 carried two point mutations (G62R and W700stop) at the N- and C-terminal ends of the coding sequence, respectively. Here, we further characterized parc6-5 and other parc6 mutant alleles, and showed that PARC6 plays a critical role in plastid morphogenesis in all cell types of the leaf epidermis: pavement cells, trichome cells, and guard cells. Transient expression of PARC6 transit peptide (TP) fused to the green fluorescent protein (GFP) in plant cells showed that the G62R mutation has no or little effect on the TP activity of the PARC6 N-terminal region. Then, plastid morphology was microscopically analyzed in the leaf epidermis of wild-type (WT) and parc6 mutants (parc6-1, parc6-3, parc6-4 and parc6-5) with the aid of stroma-targeted fluorescent proteins. In parc6 pavement cells, plastids often assumed aberrant grape-like morphology, similar to those in severe plastid division mutants, atminE1, and arc6. In parc6 trichome cells, plastids exhibited extreme grape-like aggregations, without the production of giant plastids (>6 µm diameter), as a general phenotype. In parc6 guard cells, plastids exhibited a variety of abnormal phenotypes, including reduced number, enlarged size, and activated stromules, similar to those in atminE1 and arc6 guard cells. Nevertheless, unlike atminE1 and arc6, parc6 exhibited a low number of mini-chloroplasts (< 2 µm diameter) and rarely produced chloroplast-deficient guard cells. Importantly, unlike parc6, the chloroplast division site mutant arc11 exhibited WT-like plastid phenotypes in trichome and guard cells. Finally, observation of parc6 complementation lines expressing a functional PARC6-GFP protein indicated that PARC6-GFP formed a ring-like structure in both constricting and non-constricting chloroplasts, and that PARC6 dynamically changes its configuration during the process of chloroplast division.

LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa.

PURPOSE: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora. MATERIALS AND METHODS: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined. RESULTS: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET). CONCLUSIONS: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

An early-flowering einkorn wheat mutant with deletions of PHYTOCLOCK 1/LUX ARRHYTHMO and VERNALIZATION 2 exhibits a high level of VERNALIZATION 1 expression induced by vernalization.

Using heavy-ion beam mutagenesis of Triticum monococcum strain KU104-1, we identified a mutant that shows extra early-flowering; it was named extra early-flowering 3 (exe3). Here, we carried out expression analyses of clock-related genes, clock downstream genes and photoperiod pathway genes, and found that the clock component gene PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX) was not expressed in exe3 mutant plants. A PCR analysis of DNA markers indicated that the exe3 mutant had a deletion of wheat PCL1/LUX (WPCL1), and that the WPCL1 deletion was correlated with the mutant phenotype in the segregation line. We confirmed that the original strain KU104-1 carried a mutation that produced a null allele of a flowering repressor gene VERNALIZATION 2 (VRN2). As a result, the exe3 mutant has both WPCL1 and VRN2 loss-of-function mutations. Analysis of plant development in a growth chamber showed that vernalization treatment accelerated flowering time in the exe3 mutant under short day (SD) as well as long day (LD) conditions, and the early-flowering phenotype was correlated with the earlier up-regulation of VRN1. The deletion of WPCL1 affects the SD-specific expression patterns of some clock-related genes, clock downstream genes and photoperiod pathway genes, suggesting that the exe3 mutant causes a disordered SD response. The present study indicates that VRN1 expression is associated with the biological clock and the VRN1 up-regulation is not influenced by the presence or absence of VRN2.